Quarterly Reviews of Biophysics

In this issue, we introduce a new format. To meet reader demand, QRB will augment its long-standing commitment to Reviews with Essays. Essays will be brief, timely commentaries considering one or more of the following: a new idea, a new argument about an old idea, the answer to an outstanding question, a new method, a new interpretation, or a subject otherwise of importance to biophysicists. Whereas Reviews are intellectually rigorous reports that stand as definitive statements on a subject for many years, Essays will seek to apply the same high standard to highlighting a moment in time, to show us where we stand and, so, to infer our trajectory. Essays will appear as deemed appropriate; this inaugural year will be replete with an Essay each quarter. The Board is pleased to announce that our Essayists for 2004 will be Richard Henderson (this issue), Donald Crothers (May), Christopher Miller (August) and Brian Matthews (November).

The flash-cooling of crystals in macromolecular crystallography has become commonplace. The procedure makes it possible to collect data from much smaller specimens than was the case in the past. Also, flash-cooled crystals are much less prone to radiation damage than their room-temperature counterparts, allowing data to be accumulated over extended periods of time. Notwithstanding the attractiveness of the technique, it does have potential disadvantages. First, better methods need to be developed to prevent damage to crystals on freezing. There is also a risk that structures determined at low temperature may suggest conclusions based on aspects of the structure that are not necessarily relevant at room temperature.

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.

1. Introduction 3

2. Background 5

3. 2D crystals 7

4. 1D crystals (helical arrays) 8

5. Icosahedral single particles 8

6. Single particles with lower symmetry 9

7. Cellular and subcellular electron tomography 10

8. Conclusion and future prospects 10

9. References 11

Structural analysis by electron microscopy of biological macromolecules or macromolecular assemblies embedded in rapidly frozen, vitreous ice has made great advances during the last few years. Electron cryo-microscopy, or cryo-EM, can now be used to analyse the structures of molecules arranged in the form of two-dimensional crystals, helical arrays or as single particles with or without symmetry. Although it has been possible, using crystalline or helical specimens, to reach a resolution adequate to build atomic models (4 Å), there is every hope this will soon also be possible with single particles. Small and large single particles present different obstacles to progress.

We review recent computational advances in the study of membrane proteins, focusing on those that have at least one transmembrane helix. Transmembrane protein regions are, in many respects, easier to investigate computationally than experimentally, due to the uniformity of their structure and interactions (e.g. consisting predominately of nearly parallel helices packed together) on one hand and presenting the challenges of solubility on the other. We present the progress made on identifying and classifying membrane proteins into families, predicting their structure from amino-acid sequence patterns (using many different methods), and analyzing their interactions and packing. The total result of this work allows us for the first time to begin to think about the membrane protein interactome, the set of all interactions between distinct transmembrane helices in the lipid bilayer.

Metals are commonly found as natural constituents of proteins. Since many such metals can interact specifically with their corresponding unfolded proteins in vitro, cofactor-binding prior to polypeptide folding may be a biological path to active metalloproteins. By interacting with the unfolded polypeptide, the metal may create local structure that initiates and directs the polypeptide-folding process. Here, we review recent literature that addresses the involvement of metals in protein-folding reactions in vitro. To date, the best characterized systems are simple one such as blue-copper proteins, heme-binding proteins, iron-sulfur-cluster proteins and synthetic metallopeptides. Taken together, the available data demonstrates that metals can play diverse roles: it is clear that many cofactors bind before polypeptide folding and influence the reaction; yet, some do not bind until a well-structured active site is formed. The significance of characterizing the effects of metals on protein conformational changes is underscored by the many human diseases that are directly linked to anomalous protein–metal interactions.

1. Introduction 17

2. Dynamics of many-body systems 19

2.1 Effective dynamics of reduced systems 21

2.2 The constraint of thermodynamic equilibrium 24

2.3 Mean-field theories 25

3. Solvation free energy and electrostatics 27

3.1 Microscopic view of the Born model 27

3.2 Ion–Ion interactions in bulk solution 29

3.3 Continuum electrostatics and the PB equation 29

3.4 Limitations of continuum dielectric models 32

3.5 The dielectric barrier 33

3.6 The transmembrane potential and the PB-V equation 35

4. Statistical mechanical equilibrium theory 40

4.1 Multi-ion PMF 40

4.2 Equilibrium probabilities of occupancy 43

4.3 Coupling to the membrane potential 44

4.4 Ionic selectivity 48

4.5 Reduction to a one-dimensional (1D) free-energy profile 49

5. From MD toI–V: a practical guide 50

5.1 Extracting the essential ingredients from MD 51

5.1.1 Channel conductance from equilibrium and non-equilibrium MD 51

5.1.2 PMF techniques 52

5.1.3 Friction and diffusion coefficient techniques 53

5.1.4 About computational times 55

5.2 Ion permeation models 56

5.2.1 The 1D-NP electrodiffusion theory 56

5.2.2 Discrete-state Markov chains 57

5.2.3 The GCMC/BD algorithm 58

5.2.4 PNP electrodiffusion theory 62

6. Computational studies of ion channels 63

6.1 Computational studies of gA 65

6.1.1 Free-energy surface for K+ permeation 66

6.1.2 Mean-force decomposition 69

6.1.3 Cation-binding sites 69

6.1.4 Channel conductance 70

6.1.5 Selectivity 72

6.2 Computational studies of KcsA 72

6.2.1 Multi-ion free-energy surface and cation-binding sites 73

6.2.2 Channel conductance 74

6.2.3 Mechanism of ion conduction 77

6.2.4 Selectivity 78

6.3 Computational studies of OmpF 79

6.3.1 The need to compare the different level of approximations 79

6.3.2 Equilibrium protein fluctuations and ion distribution 80

6.3.3 Non-equilibrium ion fluxes 80

6.3.4 Reversal potential and selectivity 84

6.4 Successes and limitations 87

6.4.1 Channel structure 87

6.4.2 Ion-binding sites 87

6.4.3 Ion conduction 88

6.4.4 Ion selectivity 89

7. Conclusion 90

8. Acknowledgments 93

9. References 93

The goal of this review is to establish a broad and rigorous theoretical framework to describe ion permeation through biological channels. This framework is developed in the context of atomic models on the basis of the statistical mechanical projection-operator formalism of Mori and Zwanzig. The review is divided into two main parts. The first part introduces the fundamental concepts needed to construct a hierarchy of dynamical models at different level of approximation. In particular, the potential of mean force (PMF) as a configuration-dependent free energy is introduced, and its significance concerning equilibrium and non-equilibrium phenomena is discussed. In addition, fundamental aspects of membrane electrostatics, with a particular emphasis on the influence of the transmembrane potential, as well as important computational techniques for extracting essential information from all-atom molecular dynamics (MD) simulations are described and discussed. The first part of the review provides a theoretical formalism to ‘translate’ the information from the atomic structure into the familiar language of phenomenological models of ion permeation. The second part is aimed at reviewing and contrasting results obtained in recent computational studies of three very different channels; the gramicidin A (gA) channel, which is a narrow one-ion pore (at moderate concentration), the KcsA channel from Streptomyces lividans, which is a narrow multi-ion pore, and the outer membrane matrix porin F (OmpF) from Escherichia coli, which is a trimer of three β-barrel subunits each forming wide aqueous multi-ion pores. Comparison with experiments demonstrates that current computational models are approaching semi-quantitative accuracy and are able to provide significant insight into the microscopic mechanisms of ion conduction and selectivity. We conclude that all-atom MD with explicit water molecules can represent important structural features of complex biological channels accurately, including such features as the location of ion-binding sites along the permeation pathway. We finally discuss the broader issue of the validity of ion permeation models and an outlook to the future.

Volume 37 Number 2 (May 2004), pp. 147–195

Cell-penetrating peptides: from inception to application

Due to a correction error at the Press stage the title of this paper was incorrectly published with the word ‘small’ included. The correct title is given above.

The publisher apologizes to the authors and readers for this error.

Despite continuing advances in the development of macromolecules, including peptides, proteins, and oligonucleotides, for therapeutic purposes, the successful application of these hydrophilic molecules has so far been hampered by their inability to efficiently traverse the cellular plasma membrane. The discovery of a class of peptides (cell-penetrating peptides, CPPs) with the ability to mediate the non-invasive and efficient import of a whole host of cargoes, both in vitro and in vivo, has provided a new means by which the problem associated with cellular delivery can be circumvented. A complete understanding of the translocation mechanism(s) of CPPs has so far proven elusive. Initial studies indicated an ATP-independent, non-endocytotic mechanism, dependent on direct peptide–membrane interactions, making it an enticing challenge from a biophysical point of view. However, recent evidence cast doubt on many of the earlier results, and led to a re-evaluation of the translocation mechanism of CPPs. In this review a brief history of the field will be given, followed by an introduction to some of the better known and more widely used CPPs, including some of their current applications, and finally a discussion of the translocation mechanism(s) and the controversies surrounding it.